The overall goal of this project is to increase cure of childhood acute lymphoblastic leukemia (ALL) by selectively modulating in leukemic blasts the uptake and metabolism of methotrexate (MTX), a drug key in curing this disease. Our approach is based on our unique finding that exogenous 5-amino-4-imidazole-carboxamide riboside (Z) potentiates uptake, metabolism, and cytotoxic potency of MTX in a preclinical childhood ALL model. Since Z has been used in clinical trials for treating cardiac ischemia, our preclinical studies may be readily translatable into a pediatric clinical trial. Critical to appropriate use of Z as a potentiator is an understanding of its mechanism, which will be elucidated through five Specific Aims: 1) Characterize Z potentiation of MTX uptake and metabolism in a cell culture model of childhood ALL. Our results show that Z potentiates MTX accumulation and metabolism to MTX polyglutamates primarily via a previously undiscovered enhancement in activity of the reduced folate carrier (RFC), which also transports MTX. Z potentiation of MTX uptake by the RFC and the role of other factors in potentiation of MTX polyglutamate accumulation must be fully defined. 2) Determine the role of dihydrofolate reductase (DHFR) inhibition in Z potentiation of MTX uptake. 3) Determine whether specific Z metabolism is required for potentiation of MTX uptake. A requirement for Z transport and metabolism will be explored by using inhibitors of, and/or using cell lines with mutations in, specific nucleoside transport or metabolizing enzymes and by using direct quantitation of potential metabolites of radiolabeled Z. 4) Determine the biochemical mechanisms that transduce the Z signal into increased RFC activity. Since new protein synthesis is not required (Results), Z could increase RFC activity by inducing a post-translational modification or allosterically. Both classes of mechanism will be explored. 5) Define the clinical potential of Z potentiation by determining the dose, schedule, and therapeutic efficacy of MTX+Z combinations in in vivo preclinical models of childhood ALL. If successful, this research could provide a way to increase the effectiveness of methotrexate, a key drug used in treating childhood acute lymphoblastic leukemia, and thus increase the cure rate in this disease.